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

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 .

Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients.

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of ≥300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Na+-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.NEW & NOTEWORTHY The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation, and apical membrane attack in kidney transplant recipients. C3dg and C5b-9-associated C9 neoantigen associate with proximal tubular apical membranes as demonstrated in urine extracellular vesicles. The discovery suggests intratubular complement as a mediator between proteinuria and progressive kidney damage. Inhibitors of soluble and/or luminal complement activation with access to the tubular lumen may be beneficial.

The epithelial Na+ channel α- and γ-subunits are cleaved at predicted furin-cleavage sites, glycosylated and membrane associated in human kidney.

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.

Dietary Na+ intake in healthy humans changes the urine extracellular vesicle prostasin abundance while the vesicle excretion rate, NCC, and ENaC are not altered.

Low Na+ intake activates aldosterone signaling, which increases renal Na+ reabsorption through increased apical activity of the NaCl cotransporter (NCC) and the epithelial Na+ channel (ENaC). Na+ transporter proteins are excreted in urine as an integral part of cell-derived extracellular vesicles (uEVs). It was hypothesized that Na+ transport protein levels in uEVs from healthy humans reflect their physiological regulation by aldosterone. Urine and plasma samples from 10 healthy men (median age: 22.8 yr) were collected after 5 days on a low-Na+ (70 mmol/day) diet and 5 days on a high-Na+ (250 mmol/day) diet. uEVs were isolated by ultracentrifugation and analyzed by Western blot analysis for EV markers (CD9, CD63, and ALIX), transport proteins (Na+-K+-ATPase α1-subunit, NCC, ENaC α- and γ-subunits, and aquaporin 2), and the ENaC-cleaving protease prostasin. Plasma renin and aldosterone concentrations increased during the low-Na+ diet. uEV size and concentration were not different between diets by tunable resistive pulse sensing. EV markers ALIX and CD9 increased with the low-Na+ diet, whereas CD63 and aquaporin 2 excretion were unchanged. Full-length ENaC γ-subunits were generally not detectable in uEVs, whereas ENaC α-subunits, NCC, and phosphorylated NCC were consistently detected but not changed by Na+ intake. Prostasin increased with low Na+ in uEVs. uEV excretion of transporters was not correlated with blood pressure, urinary Na+ and K+ excretion, plasma renin, or aldosterone. In conclusion, apical Na+ transporter proteins and proteases were excreted in uEVs, and while the excretion rate and size of uEVs were not affected, EV markers and prostasin increased in response to the low-Na+ diet.

Hydronephrosis is associated with elevated plasmin in urine in pediatric patients and rats and changes in NCC and γ-ENaC abundance in rat kidney.

Obstruction of urine flow at the level of the pelvo-ureteric junction (UPJO) and subsequent development of hydronephrosis is one of the most common congenital renal malformations. UPJO is associated with development of salt-sensitive hypertension, which is set by the obstructed kidney, and with a stimulated renin-angiotensin-aldosterone system (RAAS) in rodent models. This study aimed at investigating the hypothesis that 1) in pediatric patients with UPJO the RAAS is activated before surgical relief of the obstruction; 2) in rats with UPJO the RAAS activation is reflected by increased abundance of renal aldosterone-stimulated Na transporters; and 3) the injured UPJO kidney allows aberrant filtration of plasminogen, leading to proteolytic activation of the epithelial Na channel γ-subunit (γ-ENaC). Hydronephrosis resulting from UPJO in pediatric patients and rats was associated with increased urinary plasminogen-to-creatinine ratio. In pediatric patients, plasma renin, angiotensin II, urine and plasma aldosterone, and urine soluble prorenin receptor did not differ significantly before or after surgery, or compared with controls. Increased plasmin-to-plasminogen ratio was seen in UPJO rats. Intact γ-ENaC abundance was not changed in UPJO kidney, whereas low-molecular cleavage product abundance increased. The Na-Cl cotransporter displayed significantly lower abundance in the UPJO kidney compared with the nonobstructed contralateral kidney. The Na-K-ATPase α-subunit was unaltered. Treatment with an angiotensin-converting enzyme inhibitor (8 days, captopril) significantly lowered blood pressure in UPJO rats. It is concluded that the RAAS contributes to hypertension following partial obstruction of urine flow at the pelvo-ureteric junction with potential contribution from proteolytic activation of ENaC.

Albuminuria in kidney transplant recipients is associated with increased urinary serine proteases and activation of the epithelial sodium channel.

Albuminuria predicts adverse renal outcome in kidney transplant recipients. The present study addressed the hypothesis that albuminuria is associated with increased urine serine proteases with the ability to activate the epithelial sodium channel (ENaC) and with greater extracellular volume and higher blood pressure. In a cross-sectional design, kidney transplant recipients with ( n = 18) and without ( n = 19) albuminuria were included for office blood pressure measurements, estimation of volume status by bioimpedance, and collection of spot urine and plasma samples. Urine was analyzed for serine proteases and for the ability to activate ENaC current in vitro. Urine exosome protein was immunoblotted for prostasin and γ-ENaC protein. In the present study, it was found that, compared with nonalbuminuria (8.8 mg/g creatinine), albuminuric (1,722 mg/g creatinine) kidney transplant recipients had a higher systolic and diastolic blood pressure, despite receiving significantly more antihypertensives, and a greater urinary total plasminogen, active plasmin, active urokinase-type plasminogen activator, and prostasin protein abundance, which correlated significantly with u-albumin. Fluid overload correlated with systolic blood pressure, urinary albumin/creatinine, and plasminogen/creatinine. Urine from albuminuric kidney transplant recipients evoked a greater amiloride- and aprotinin-sensitive inward current in single collecting duct cells (murine cell line M1). γENaC subunits at 50 and 75 kDa showed increased abundance in urine exosomes from albuminuric kidney transplant recipients when compared with controls. These findings show that albuminuria in kidney transplant recipients is associated with hypertension, ability of urine to proteolytically activate ENaC current, and increased abundance of γENaC. ENaC activity could contribute to hypertension and adverse outcome in posttransplant proteinuria.

Physiology and pathophysiology of the plasminogen system in the kidney.

The plasminogen system is important for fibrinolysis in addition to tissue remodeling and inflammation with significance for kidney disease. The system consists of the circulating zymogen plasminogen (Plg) and the tissue- and urokinase-type plasminogen activators, tPA and uPA, expressed in the glomeruli, endothelium and tubular epithelium, respectively, and the inhibitors α2-antiplasmin and plasminogen activator inhibitor-type1, PAI-1. Plasminogen is activated by surface receptors, some with renal expression: urokinase-type plasminogen activator receptor (uPAR), plasminogen receptor KT (Plg-RKT), and tPA, most evident in the endothelium. Plasmin may exert effects through protease-activated receptors, PARs, expressed in the kidney. Deletion of plasminogen system component genes confers no major developmental or renal phenotypes in normal mice. In glomerular injury and renal interstitial fibrosis, deletion of various components, notably Plg, uPA, PAI, and uPAR is associated with protection suggesting a disease promoting effect of plasmin, in some cases exerted through PAR1 receptor activation. Plasminogen and uPA are aberrantly filtrated across the glomerular barrier in proteinuria, and plasminogen is activated in the tubular fluid. In the tubular fluid, plasmin may activate proteolytically the epithelial sodium channel (ENaC) and inhibit the apical calcium transporter transient receptor potential cation channel subfamily V member 5 (TRPV5), which could explain impaired sodium excretion and enhanced calcium excretion in proteinuria. Amiloride, a potassium-sparing diuretic, inhibits urokinase and plasmin activation in the tubular fluid and uPAR expression in vitro, which highlights new indications for an old drug. Protease inhibitors lowered blood pressure and antagonized fibrosis in salt-sensitive Dahl rats. Current knowledge indicates that the plasminogen system aggravates renal disease by direct and indirect hypertensive effects and is a promising target to antagonize disease progression.

Urine exosomes from healthy and hypertensive pregnancies display elevated level of α-subunit and cleaved α- and γ-subunits of the epithelial sodium channel-ENaC.

Preeclampsia is characterized by hypertension, proteinuria, suppression of plasma renin-angiotensin-aldosterone, and impaired urine sodium excretion. Aberrantly filtered plasmin in urine may activate proteolytically the γ-subunit of the epithelial sodium channel (ENaC) and promote Na+ reabsorption and urine K+ loss. Plasma and urine was sampled from patients with preeclampsia, healthy pregnant controls and non-pregnant women, and from patients with nephrostomy catheters. Aldosterone concentration, urine plasminogen, and protein were determined. Exosomes were isolated by ultracentrifugation. Immunoblotting was used to detect exosome markers; γ-ENaC (two different epitopes within the inhibitory peptide tract), α-ENaC, and renal outer medullary K-channel (ROMK) and compared with human kidney cortex homogenate. Urine total plasmin(ogen) was significantly increased in preeclampsia, plasma and urine aldosterone was higher in pregnancy compared to non-pregnancy, and the urine Na/K ratio was lower in preeclampsia compared to healthy pregnancy. Exosome markers ALIX and AQP-2 were stably associated with exosomes across groups. Exosomal α-ENaC-subunit migrated at 75 kDa and dominantly at 50 kDa and was significantly elevated in pregnancy. In human kidney cortex tissue and two of four pelvis catheter urine, ~90-100 kDa full-length γ-ENaC was detected while no full-length γ-ENaC but 75, 60, and 37 kDa variants dominated in voided urine exosomes. There was no difference in γ-ENaC protein abundances between healthy pregnancy and preeclampsia. ROMK was detected inconsistently in urine exosomes. Pregnancy and preeclampsia were associated with increased abundance of furin-cleaved α-ENaC subunit while γ-subunit appeared predominantly in cleaved form independently of conditions and with a significant contribution from post-renal cleavage.

The epithelial sodium channel γ-subunit is processed proteolytically in human kidney.

The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the γ-subunit and putative release of a 43-amino acid inhibitory tract from the γ-subunit ectodomain. We hypothesized that proteolytic processing of γENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved γENaC or the furin-cleaved-to-full-length ratio of γENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved γENaC, with no significant change in the furin-cleaved-to-full-length γENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length γENaC by mAbinhibit. Prostasin/kallikrein-cleaved γENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney γENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within γENaC.

Mannan-binding lectin serine protease-2 (MASP-2) in human kidney and its relevance for proteolytic activation of the epithelial sodium channel.

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+ intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+ diet and the ratio correlated with changes in aldosterone and fractional Na+ excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+ intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.