Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria

Abstract

Significance statement: Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function.

Background: Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor.

Methods: Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed.

Results: The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly.

Conclusions: In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction.

Clinical trial registry name and registration number: Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .

Graphical abstract

Figure 1

C3 and C5 are cleaved via uPA-plasminogen cascade in vitro. (A) Active plasmin induces concentration-dependent activation of C3. Nonreduced samples are shown in lanes 1–4. Corresponding samples display a migratory shift from α- to α′-chain under reducing conditions in lanes 5–8, indicating separation of C3a from the α-chain. (B) Plasmin cleaves and activates C5 generating C5b, migrating at approximately 190 and 180 kDa, respectively, under nonreducing conditions (lanes 1–4). Reduced samples are shown in lanes 5–8 and indicate that plasmin further cleaves the C5 α-chain, but not the β-chain, to smaller fragments in a concentration-dependent manner. (C) Activation of C3 is inhibited by increasing concentrations of amiloride. Nonreduced (lanes 1–6) and corresponding reduced samples (lanes 7–12) are shown. SDS–PAGE separated proteins were visualized with Coomassie staining. Experiments were performed four times and representative gels are shown. L, ladder; Pl, plasmin; Plg, plasminogen; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; uPA, urokinase-type plasminogen activator.

Figure 2

Amiloride inhibits uPA-mediated generation of C3a and C5a in vitro. uPA was preincubated for 10 minutes with different concentrations of amiloride or with TBS. Next, pure plasminogen and C3, or C5, were added and incubated at 37°C. The mixture was subsequently subjected to immunoblotting. (A) Immunoblot for C3a (predicted migrating at 9 kDa); lanes 1–4: uPA-plasminogen-C3 with amiloride 4 µM–4 mM, lane 5: full reaction without amiloride, lane 6: plasminogen with C3 (negative control), and lane 7: pure C3a (100 ng, positive control). (B) Immunoblot for C5a (predicted migration at 9 kDa but appears glycosylated at 15 kDa); lanes 1–4: uPA-plasminogen-C5 with amiloride 4 µM–4 mM, lane 5: full reaction without amiloride, lane 6: plasminogen with C5 (negative control), and lane 7: pure C5a (50 ng, positive control). Inhibition of the uPA-plasminogen C3/C5 reactions by amiloride was also assessed by specific ELISA for (C) C3a and (D) C5a. Samples were run in two-fold serial dilution. Representative experiments of n=2–3 are shown. ODF, optical density fluorescence; TBS, Tris buffered saline.

Figure 3

Urine from healthy humans promotes anaphylatoxin generation from C3 and C5 only in the presence of exogenous plasminogen. ELISA for C3a showing the effect of (A) ex vivo addition of C3 (10 µg/ml) to normal human urine in the presence or absence of plasminogen (40 µg/ml). (B) C3a generation from C3 in the presence of exogenous plasminogen in urine preincubated with amiloride (500 µM), aprotinin (500 KIU/ml), or EDTA (20 mM) (experiment n=5 run in parallel). ELISA showing C5a generation from (C) C5 (10 µg/ml) in urine when increasing amounts of plasminogen (0.05–50 µg/ml) were added and (D) in urine preincubated with amiloride, aprotinin, or EDTA when C5 (10 µg/ml) and plasminogen (50 µg/ml) were added (n=4 per group). (D) C5a generation from C5 in the presence of exogenous plasminogen in urine preincubated with amiloride (500 µM), aprotinin (500 KIU/ml), or EDTA (20 mM, experiment n=4 run in parallel). Means with scatter are presented in (B) and (D). *P < 0.05; ***P < 0.001.

Figure 4

Urine excretion of anaphylatoxins in humans relates to proteinuria. (A) C3a/creatinine ratio in urine from healthy controls (n=6), in patients with albuminuria (n=10), and KTRs with (n=7) or without (n=7) clinically significant albuminuria. (B) Corresponding plasma concentrations for KTRs. (C) Urine C3a and albumin concentrations from pooled individuals with albuminuria (n=17) were related in a log–log linear fashion: slope with 95% CI: 2.4 (1.7 to 3.8). (D) C5a/creatinine ratio for healthy controls, patients with albuminuria, and KTRs with or without albuminuria. (E) C5a plasma concentration in KTRs. (F) Log–log linear relationship between pooled urine C5a and albumin concentrations: slope 2.3 (1.6 to 4.0). Medians with scatter are presented in (A), (B), (D), and (E). Log–log lines are plotted in (C) and (F). Unpaired t test *P < 0.05; **P < 0.01; ****P < 0.0001. CI, confidence interval; KTR, kidney transplant recipient; UACR, urine albumin-creatinine ratio.

Figure 5

Effect of high-dose amiloride for 2 days in patients with type 1 diabetes with and without diabetic nephropathy (DN). Graphs show (A) urinary C3dg/creatinine ratio and (B) urinary C9 neoantigen (C9 neo)/creatinine ratio for controls and patients with DN. In the DN group, only patients with urine complement within measurable range were included in the statistical analysis and plotted (C3dg: n=5 of 15, C9 neoantigen: n=10 of 15). All controls are shown. (C) Corresponding plasma concentrations of C3dg and (D) C9 neoantigen. Paired plasma samples from four controls and four cases were missing from the biobank and were thus not measured. *P < 0.05.

Figure 6

Urine anaphylatoxins in conditional podocin KO mice with severe proteinuria. Twenty-four–hour urine excretion of anaphylatoxins was measured in two series of mice with conditional KO of podocin, amiloride for 4 days or vehicle, or an anti-uPA–inhibiting antibody or a control antibody. A baseline urine sample was collected on day −7, and Cre induction with tamoxifen was performed on days −4 to 0. Mice in the amiloride trial were divided in two groups receiving either amiloride (■ and dashed line) or vehicle (● and full line) by two daily i.p. injections and kept in metabolic cages from days 10–20 during the experiment. Amiloride was administered in a step-up fashion: 2.5 mg/kg on days 13 and 14 and 10 mg/kg on days 15 and 16 (shaded area in A and B), followed by a 3-day washout period. Twenty-four–hour urine excretion rates are shown for (A) C3a and (B) C5a, in amiloride- (n=7) and vehicle- (n=7) treated podocin KO mice. As published earlier, albumin excretion did not change between groups in amiloride-treated animals. In the second series, inhibiting anti-uPA antibodies (120 mg/kg per day) or a similar dose of isotype-matched control IgG antibodies were injected i.p. from days 9–20 (shaded areas in C–E). Urine excretion rates are presented for (C) C3a, (D) C5a, and (E) albumin in anti-uPA-mice (□ and dashed line, n=5) and isotype control antibody-mice (○ and full line, n=4). Urine was collected directly into tubes containing protease inhibitor cocktail, and excretion rates are normalized to body weight measured daily. Kidney tissue protein concentrations are shown for (F) C3a and (G) C5a. Data are presented as mean with SEM in (A–G) and mean with SD in (F) and (G). *P < 0.05 compared with vehicle by Student's t test. #P < 0.05 by 2w-ANOVA between groups. Mixed effects analyses with missing values were also tested and produced similar results. 2w-ANOVA, two-way ANOVA; i.p., intraperitoneal; KO, knockout; ns, not significant.

Figure 7

Kidney inflammation and fibrosis in anti-uPA–treated podocin KO mice. Kidneys were harvested from podocin KO mice after 11 days of treatment with either anti-uPA antibodies or isotype control antibodies, and 24-hour urine samples from baseline and days 12, 16, and 19 were analyzed. (A) Immunoblots for oxidative stress markers HO-1 (expected migration 33 kDa and approximately 25 kDa in truncated form), SOD1 (20 kDa), and SOD2 (27 kDa) and α-tubulin. (B) Semiquantified data from immunoblots for HO-1, SOD1, SOD2, and NLRP3 normalized to α-tubulin. (C) Immunoblots for E-cad (expected migration 110 kDa), αSMA (expected migration 42 kDa), collagen I (expected migration of procollagen I approximately 250 kDa; collagen I 138 kDa but observed approximately 55 kDa, weak bands also appear at approximately 150 and approximately 100 kDa), and α-tubulin (expected migration 50 kDa). Wild-type mouse kidney tissue was used as negative control (Neg), and kidney tissue from a wild-type mouse subjected to unilateral ureter obstruction was used as positive control (Pos). (D) Semiquantified data from immunoblots for E-cadherin, αSMA, and collagen I normalized to α-tubulin. (E) Urine excretion rate for KIM-1 in anti-uPA–treated mice (□ and dashed line, n=5) and isotype control antibody–treated mice (○ and full line, n=4). (F) Kidney tissue levels of KIM-1 protein normalized to kidney weight after 11 days of treatment. Data are presented as mean with SD. *P < 0.05. αSMA, α-smooth muscle actin; E-cad, E-cadherin; HO-1, heme oxygenase 1; KIM-1, kidney injury molecule 1; NLRP3, NOD-, LRR-, and pyrin domain-containing protein 3; SOD1, super oxide dismutase 1; SOD2, super oxide dismutase 1.