Urinary Plasminogen as a Marker of Disease Progression in Human Glomerular Disease.

RATIONALE & OBJECTIVE: Glomerular disorders have a highly variable clinical course, and biomarkers that reflect the molecular mechanisms underlying their progression are needed. Based on our previous work identifying plasminogen as a direct cause of podocyte injury, we designed this study to test the association between urine plasmin(ogen) (ie, plasmin and its precursor plasminogen) and end-stage kidney disease (ESKD). STUDY DESIGN: Multicenter cohort study. SETTING & PARTICIPANTS: 1,010 patients enrolled in the CureGN Cohort with biopsy-proven glomerular disease (focal segmental glomerulosclerosis, membranous nephropathy, and immunoglobulin A nephropathy). PREDICTORS: The main predictor was urine plasmin(ogen) at baseline. Levels were measured by an electrochemiluminescent immunoassay developed de novo. Traditional clinical and analytical characteristics were used for adjustment. The ratio of urine plasmin(ogen)/expected plasmin(ogen) was evaluated as a predictor in a separate model. OUTCOME: Progression to ESKD. ANALYTICAL APPROACH: Cox regression was used to examine the association between urinary plasmin(ogen) and time to ESKD. Urinary markers were log2 transformed to approximate normal distribution and normalized to urinary creatinine (Log2uPlasminogen/cr, Log2 urinary protein/cr [UPCR]). Expected plasmin(ogen) was calculated by multiple linear regression. RESULTS: Adjusted Log2uPlasminogen/cr was significantly associated with ESKD (HR per doubling Log2 uPlasminogen/cr 1.31 [95% CI, 1.22-1.40], P<0.001). Comparison of the predictive performance of the models including Log2 uPlasminogen/cr, Log2 UPCR, or both markers showed the plasmin(ogen) model superiority. The ratio of measured/expected urine plasmin(ogen) was independently associated with ESKD: HR, 0.41 (95% CI, 0.22-0.77) if ratio<0.8 and HR 2.42 (95% CI, 1.54-3.78) if ratio>1.1 (compared with ratio between 0.8 and 1.1). LIMITATIONS: Single plasmin(ogen) determination does not allow for the study of changes over time. The use of a cohort of mostly white patients and the restriction to patients with 3 glomerular disorders limits the external validity of our analysis. CONCLUSIONS: Urinary plasmin(ogen) and the ratio of measured/expected plasmin(ogen) are independently associated with ESKD in a cohort of patients with glomerular disease. Taken together with our previous experimental findings, urinary plasmin(ogen) could be a useful biomarker in prognostic decision making and a target for the development of novel therapies in patients with proteinuria and glomerular disease. PLAIN-LANGUAGE SUMMARY: Glomerular diseases are an important cause of morbidity and mortality in patients of all ages. Knowing the individual risk of progression to dialysis or transplantation would help to plan the follow-up and treatment of these patients. Our work studies the usefulness of urinary plasminogen as a marker of progression in this context, since previous studies indicate that plasminogen may be involved in the mechanisms responsible for the progression of these disorders. Our work in a sample of 1,010 patients with glomerular disease demonstrates that urinary plasminogen (as well as the ratio of measured to expected plasminogen) is associated with the risk of progression to end-stage kidney disease. Urine plasminogen exhibited good performance and, if further validated, could enable risk stratification for timely interventions in patients with proteinuria and glomerular disease.

Urinary pro-thrombotic, anti-thrombotic, and fibrinolytic molecules as biomarkers of lupus nephritis.

OBJECTIVE: This study evaluates the utility of urinary pro-thrombotic molecules such as tissue factor (TF), anti-thrombotic molecules such as tissue factor pathway inhibitor (TFPI), and fibrinolytic molecules such as plasmin and d-dimer as biomarkers of lupus nephritis (LN). METHODS: Urine samples from 113 biopsy-proven LN patients (89 active LN and 24 inactive LN), 45 chronic kidney disease patients, and 41 healthy controls were examined for d-dimer, plasmin, TF, and TFPI levels by ELISA. The area under the receiver operating characteristic curve (AUC) analysis, multivariate regression analysis, and Bayesian network analysis were performed to assess the diagnostic value of the assayed molecules in LN. RESULTS: Although urinary d-dimer, plasmin, TF, and TFPI were all elevated in active LN compared to all control groups, and correlated with rSLEDAI and SLICC RAS disease activity indices, urine plasmin emerged as the strongest independent predictor of eGFR and renal disease status, by multivariate regression analysis and Bayesian network analysis. Whereas urine plasmin discriminated active LN from inactive disease with an AUC of 0.84, the combination of urine plasmin and TFPI discriminated ALN from ILN with an AUC of 0.86, with both surpassing the specificity and positive predictive value of traditional markers such as anti-dsDNA and complement C3. CONCLUSION: Both thrombogenic and thrombolytic cascades appear to be upregulated in lupus nephritis, with proteins from both cascades appearing in the urine. Of the coagulation cascade proteins surveyed, urine plasmin emerges as the strongest predictor of eGFR and clinical renal disease in patients with LN.

Association of Urinary Plasminogen-Plasmin with Edema and Epithelial Sodium Channel Activation in Patients with Nephrotic Syndrome.

BACKGROUND: Previous animal experiments and small human studies suggest that urinary plasmin can activate the epithelial sodium channel (ENaC) and contribute to sodium retention in nephrotic syndrome (NS), but this however is not well studied in clinical settings, and its relevance to edema formation is not well characterized in humans. We have investigated the association between urinary plasmin and clinical phenotypes in a large group of patients with NS from multiple etiologies, aiming to assess the role of urinary plasmin in sodium handling and edema formation. METHODS: Two hundred and three NS patients with urine and blood samples were divided into mild and severe symptom groups based on their edema severity. Twenty six of them had serial samples collected during the course of immunosuppressive therapy. The plasminogen-plasmin level and other key parameters were assayed, and their association with clinical manifestations were analyzed. RESULTS: One hundred and one of the 203 patients had renal biopsies performed, the results of which had included all the common types of primary NS and various types of secondary NS. Quantitative comparison and multivariate logistic regression analysis identified urinary plasminogen-plasmin to creatinine ratio (uPLG-PL/C), serum albumin, D-Dimer, and cardiac dysfunction history, but not albuminuria or 24-h urine protein, as independent risk factors for edema (p < 0.01). In patients who were treated and had serial samples, a decrease in uPLG-PL/C was identified as an independent influencing factor of edema remission (p < 0.01). Finally, the urinary fractional excretion of sodium (FENa) in patients was inversely correlated with the fractional excretion of potassium (FEK; p< 0.001), and FEK/FENa ratio was positively correlated with uPLG-PL/C (p < 0.001), suggesting a close association between uPLG-PL and ENaC activation. CONCLUSIONS: Our study identifies uPLG-PL abundance as an independent influencing factor of edema in adult NS patients, and supports the conclusion that plasmin-dependent ENaC activation is an important pathophysiological mechanism of sodium retention and edema formation in humans with NS.

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.

Urine albumin is a superior predictor of preeclampsia compared to urine plasminogen in type I diabetes patients.

Pregnant women with type I diabetes mellitus (T1DM) are at increased risk of developing preeclampsia (PE). Plasminogen is aberrantly filtrated from plasma into tubular fluid in PE patients and activated to plasmin. Plasmin activates the epithelial sodium channel in the collecting ducts potentially causing impaired sodium excretion, suppression of the renin-angiotensin-aldosterone system, and hypertension in PE. The objective of the study was to test whether urinary total plasmin(ogen)/creatinine ratio and plasma concentration of aldosterone were better predictors of PE in pregnant women with T1DM compared with urine albumin and haemoglobin A1C. The design was a longitudinal observational study of 88 pregnant T1DM patients at 2 Danish centers. Spot urine- and blood samples were collected at gestational weeks 12, 20, 28, 32, and 36. U-plasmin(ogen)/creatinine ratio increased during pregnancy. In gestational week 36, the ratio was significantly increased in the T1DM patients developing PE (P < .05). P-aldosterone was significantly increased in gestational week 20 in the group developing PE (P < .05). U-albumin/creatinine ratio was significantly increased and predicted PE at all tested gestational ages. U-albumin/creatinine ratio had a stronger association with the development of PE compared to u-total plasmin(ogen)/creatinine ratio and p-aldosterone. The positive association between u-total plasmin(ogen) and development of PE late in pregnancy is compatible with involvement in PE pathophysiology. The significance of albumin in urine emphasizes the importance of preventing renal complications when planning pregnancy in patients with type I diabetes.

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.

Significant natriuretic and antihypertensive action of the epithelial sodium channel blocker amiloride in diabetic patients with and without nephropathy.

OBJECTIVE: Diabetic nephropathy is associated with aberrant glomerular filtration of serine proteases. The study was designed to test the hypothesis that the epithelial sodium channel is activated proteolytically by urine plasmin in diabetic nephropathy and mediates renal sodium retention. METHODS: In an open-label intervention study on type 1 diabetes patients on standardized NaCl intake (200 mmol/day) with (n = 15) and without diabetic nephropathy (control, n = 12), urinary Na excretion in response to oral amiloride (20 or 40 mg/day for 2 days) was compared. RESULTS: A total of 27 patients completed the study and nine diabetic nephropathy and eight control study participants were compliant (24-h urine Na excretion of 200 mmol ±â€Š30%). Amiloride increased significantly total and fractional Na excretion in both groups. Total natriuresis and weight loss were significantly larger in the control group compared with diabetic nephropathy at day 1 of amiloride, whereas fractional Na excretion did not differ. Amiloride intervention increased plasma renin concentration only in diabetic nephropathy group; it reduced SBP in both groups, whereas DBP was reduced in diabetic nephropathy group only. Albuminuria was reduced significantly by amiloride in diabetic nephropathy group. Urine total amiloride concentration was not different between groups (12 ±â€Š1 and 16 ±â€Š1 µmol/l, respectively). Urine total plasminogen and active plasmin were reduced after amiloride in diabetic nephropathy. CONCLUSION: Amiloride increased renal Na excretion, reduced blood pressure, albuminuria, and total and active plasmin in urine. It is concluded that epithelial sodium channel is an attractive target to attain blood pressure control in long-term type I diabetes with no enhanced activity associated with nephropathy.

Association of Plasminuria with Overhydration in Patients with CKD.

BACKGROUND AND OBJECTIVES: Hypervolemia is a common feature of patients with CKD and associated with hypertension. Recent work has shown stimulation of sodium retention by urinary plasmin during nephrotic syndrome. However, it is unclear whether plasminuria plays a role in patients with stable CKD and non-nephrotic proteinuria. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this cross-sectional study, we analyzed the fluid status of 171 patients with CKD consecutively presenting to our outpatient clinic from 2012 to 2013 using bioimpedance spectroscopy (Body Composition Monitor [BCM]; Fresenius Medical Care, Germany) and its associations to the urinary excretion of plasminogen and plasmin from a spot urine sample. Two-electrode voltage clamp measurements were performed in Xenopus laevis oocytes expressing human epithelial sodium channel to investigate whether plasmin in concentrations found in urine can activate the channel. RESULTS: Overhydration >5% and overhydration >10% of the extracellular volume were found in 29% and 17% of the patients, respectively, and overhydration was associated with edema, hypertension, higher stages of CKD, and proteinuria. Proteinuria was the strongest independent predictor for overhydration (+0.58 L/1.73 m(2) per 10-fold increase; P<0.001). Urinary excretion of plasmin(ogen) quantified by ELISA correlated strongly with proteinuria (r=0.87) and overhydration (r=0.47). Using a chromogenic substrate, active plasmin was found in 44% of patients and correlated with proteinuria and overhydration. Estimated urinary plasmin concentrations were in a range sufficient to activate epithelial sodium channel currents in vitro. In multivariable analysis, urinary excretion of plasmin(ogen) was associated with overhydration similar to proteinuria. CONCLUSIONS: Hypervolemia in patients with CKD is strongly associated with proteinuria, even in the non-nephrotic range. Protein-rich urine contains high amounts of plasminogen and active plasmin, rendering plasminuria as a possible link between proteinuria and hypervolemia.

Diabetic nephropathy is associated with increased urine excretion of proteases plasmin, prostasin and urokinase and activation of amiloride-sensitive current in collecting duct cells.

BACKGROUND: Diabetic nephropathy (DN) is associated with hypertension, expanded extracellular volume and impaired renal Na(+) excretion. It was hypothesized that aberrant glomerular filtration of serine proteases in DN causes proteolytic activation of the epithelial sodium channel (ENaC) in the kidney by excision of an inhibitory peptide tract from the γ subunit. METHODS: In a cross-sectional design, urine, plasma and clinical data were collected from type 1 diabetic patients with DN (n = 19) and matched normoalbuminuric type 1 diabetics (controls, n = 20). Urine was examined for proteases by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract. RESULTS: Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion of plasmin(ogen), prostasin and urokinase that correlated directly with urine albumin. Western blotting confirmed plasmin, prostasin and urokinase in urine from the DN group predominantly. Urine from DN evoked a significantly larger amiloride-sensitive inward current in single collecting duct cells compared with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact γENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin was observed in DN samples predominantly. CONCLUSION: DN is associated with increased urinary excretion of plasmin, prostasin and urokinase and proteolytic activation of ENaC that might contribute to impaired renal Na(+) excretion and hypertension.

Plasmin in urine from patients with type 2 diabetes and treatment-resistant hypertension activates ENaC in vitro.

BACKGROUND: Aberrant filtration of plasminogen from plasma and subsequent activation to plasmin in the urinary space may activate proteolytically the epithelial sodium channel, ENaC. In conditions with chronic albuminuria, this may cause hypertension. It was hypothesized that patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension excrete plasmin(ogen) in urine in proportion to albumin and that plasmin confers to urine the ability to activate ENaC. METHOD: Patients (n = 113) with T2DM and resistant hypertension, defined as systolic blood pressure (SBP) more than 130 mmHg and/or diastolic blood pressure (DBP) more than 80 mmHg despite use of at least three drugs with one diuretic and one renin-angiotensin system inhibitor, were included. Urine was analyzed for albumin, creatinine, plasmin(ogen), protease activity, and ability to activate inward current in single collecting duct cells. RESULTS: Mean ambulatory SBP/DBP was 143 ±â€Š1/77 ±â€Š0.7 mmHg; HbA1c 7.35%; and eGFR 81.0 ml/min per 1.73 m (geometric means). Patients with microalbuminuria (39%) and macroalbuminuria (13%) displayed significantly elevated levels of urinary plasmin(ogen) normalized to urine creatinine compared with patients with normal excretion of albumin (48%). Urinary plasminogen correlated significantly to urine albumin. Western immunoblotting and gelatine zymography confirmed active plasmin in urine samples from patients with microalbuminuria and macroalbuminuria. Single collecting duct cells displayed significantly increased, amiloride-sensitive, inward current when superfused with urine from albuminuric patients compared with patients with normal albumin excretion. Urinary plasminogen/creatinine ratio correlated significantly with 24-h ambulatory blood pressure. CONCLUSION: Aberrant presence of plasmin in preurine may inappropriately activate ENaC in patients with type 2 diabetes and microalbuminuria. This may contribute to treatment-resistant hypertension.

Over- or underfill: not all nephrotic states are created equal.

Blessed were the days when it all made sense and the apparent mechanism for edema formation in nephrotic syndrome was straightforward: the kidneys lost protein in the urine, which lowered the plasma oncotic pressure. Thus, fluid leaked into the interstitium, depleting the intravascular volume with subsequent activation of renin/aldosterone and consequent avid renal sodium retention. As simple as that! Unfortunately, a number of clinical and laboratory observations have raised serious concerns about the accuracy of this "underfill" hypothesis. Instead, an "overfill" hypothesis was generated. Under this assumption, the nephrotic syndrome not only leads to urinary protein wasting, but also to primary sodium retention with consequent intravascular overfilling, with the excess fluid spilling into the flood plains of the interstitium, leading to edema. Recently, an attractive mechanism was proposed to explain this primary sodium retention: proteinuria includes plasma proteinases, such as plasmin, which activate the epithelial sodium channel in the collecting duct, ENaC. In this edition, further evidence for this hypothesis is being presented by confirming increased plasmin content in the urine of children with nephrotic syndrome and demonstrating ENaC activation. If correct, this hypothesis would provide a simple treatment for the edema: pharmacological blockade of ENaC, for instance, with amiloride. Yet, how come clinicians have not empirically discovered the presumed power of ENaC blockers in nephrotic syndrome? And why is it that some patients clearly show evidence of intravascular underfilling? The controversy of over- versus underfilling demonstrates how much we still have to learn about the pathophysiology of nephrotic syndrome.

Remission of nephrotic syndrome diminishes urinary plasmin content and abolishes activation of ENaC.

BACKGROUND: Urinary plasmin activates the epithelial Na(+) channel (ENaC) in vitro and may possibly be a mechanism of sodium retention in nephrotic syndrome (NS). This study used a paired design to test the hypothesis that remission of NS is associated with a decreased content of urinary plasmin and reduced ability of patients' urine to activate ENaC. METHODS: Samples were collected during active NS and at stable remission from 20 patients with idiopathic NS, aged 9.1 ± 3.2 years. Plasminogen-plasmin concentration was measured with an enzyme-linked immunosorbent assay. Western immunoblotting for plasminogen-plasmin was performed in paired urine samples. The patch clamp technique was used to test the ability of urine to evoke an inward current on collecting duct cells and human lymphocytes. RESULTS: The urinary plasminogen-plasmin/creatinine ratio was 226 [95 % confidence interval (CI) 130-503] µg/mmol in nephrotic urine versus 9.5 (95 % CI 8-12) µg/mmol at remission (p < 0.001). Western immunoblotting confirmed the presence of active plasmin in urine collected during active NS, while samples collected at remission were negative. Nephrotic urine generated an inward amiloride- and α2-anti-plasmin- sensitive current, whereas the observed increase in current in urine collected at remission was significantly lower (201 ± 31 vs. 29 ± 10 %; p = 0.005). CONCLUSIONS: These findings support the hypothesis that aberrantly filtered plasminogen-plasmin may contribute to ENaC activation and mediate primary renal sodium retention during active childhood NS.

Urinary plasmin inhibits TRPV5 in nephrotic-range proteinuria.

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. Patch-clamp 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.

Urinary plasmin activates collecting duct ENaC current in preeclampsia.

In nephrotic syndrome, plasminogen is aberrantly filtered from plasma to the urinary space and activated along the tubular system. In vitro, plasmin increases ENaC current by proteolytic cleavage of the γ-subunit. It was hypothesized that preeclampsia is associated with plasmin-dependent ability of tubular fluid to activate ENaC. Urine was sampled from 16 preeclamptic (PE) patients and 17 normotensive pregnant women (Ctrl). Urine was analyzed for plasmin(ogen), creatinine, albumin, aldosterone, Na(+), K(+), proteolytic activity, and for its effect on inward current in cortical collecting duct cells (M1 cells) by whole-cell patch clamp. In PE, urine plasmin(ogen): creatinine ratio was elevated 40-fold (geometric mean, 160 versus 4 µg/g; P<0.0001) and urine aldosterone: creatinine ratio was suppressed to 25% of Ctrl (geometric mean, 27 versus 109 µg/g; P<0.001). A significant negative correlation was found in PE between urinary plasmin(ogen) and aldosterone (P<0.05). In PE, proteolytic activity was detected at 90 to 75 kD by gelatin zymography in 14 of 16 patients and confirmed by serine protease assay. Immunoblotting showed active plasmin in PE urine. Whole-cell inward current increased in M1 cells on exposure to urine from PE (173±21%; n=6; P<0.001). The increase in current was abolished by amiloride (2 µmol/L; P<0.001), α(2)-antiplasmin (1 µmol/L; P<0.001), and heat denaturation (P<0.001). Preeclampsia is associated with urinary excretion of plasmin(ogen) and plasmin-dependent activation of ENaC by urine. Proteolytic activation of ENaC by plasmin may contribute to Na(+) retention and hypertension in preeclampsia.

Proteinuric diseases with sodium retention: Is plasmin the link?

1. Sodium retention in disease states characterized by proteinuria, such as nephrotic syndrome, pre-eclampsia and diabetic nephropathy, occurs through poorly understood mechanism(s). 2. In nephrotic syndrome, data from experimental and clinical studies show that the sodium retention originates in the renal cortical collecting duct and involves hyperactivity of the epithelial sodium channel (ENaC). 3. The stimulus for the increased ENaC activity does not appear to involve any of the classical sodium retaining mechanisms, such as the renin-angiotensin-aldosterone system, arginine vasopressin or the sympathetic nervous system. 4. Proteolytic processing of the extracellular domain of γENaC subunit has been shown to stimulate ENaC activity. 5. The serine protease plasmin was recently identified as an ENaC-activating protease in urine from human nephrotic patients and from the puromycin aminonucleoside (PAN) rat model of nephrotic syndrome. 6. This finding suggests that a defective glomerular filtration barrier allows filtration into the tubular fluid of substances that activate ENaC and enhance sodium reabsorption. This concept might be expanded to other disease states, such as pre-eclampsia and diabetic nephropathy, which are also characterized by proteinuria and sodium retention. 7. In this review, we will examine the evidence for a role of urinary serine protease activity in the development of sodium and water retention in diseases characterised by proteinuria with a focus on nephrotic syndrome.

Potential role of serine proteases in modulating renal sodium transport in vivo.

The maintenance of sodium (Na+) homeostasis is an essential function of the kidney. It is achieved by a variety of transport processes localized all along the highly specialised segments of the nephron. Impairment of these transport mechanisms, and thereby Na+ handling, is associated with disturbed Na+ and water balance, leading to hypertension and oedema. This review focuses on the novel regulation of sodium reabsorption by serine proteases acting along the entire nephron.

Plasmin in nephrotic urine activates the epithelial sodium channel.

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.

Elevated urinary plasmin activity resistant to alpha2-antiplasmin in acute poststreptococcal glomerulonephritis.

BACKGROUND: A pathogenic role of intraglomerular plasmin bound to nephritogenic antigen (nephritis-associated plasmin receptor, NAPlr) and resistant to physiologic inhibitors such as alpha(2)-antiplasmin (alpha(2)-AP) has recently been proposed in acute poststreptococcal glomerulonephritis (APSGN). To confirm this concept, we analysed the urinary profile of plasmin cascade in APSGN patients. METHODS: Urine samples from 10 patients with APSGN, 12 patients with IgA nephropathy (IgAN), 10 patients with streptococcal infection without nephritis (SI) and 10 healthy control subjects were analysed. The alpha(2)-AP-resistant plasmin activity was assessed by a chromogenic assay after alpha(2)-AP was added to each urine sample. Urinary plasminogen activator (PA) and plasmin were further analysed by polyacrylamide gel zymography. Urinary NAPlr was assessed by western blot analysis in selected samples. RESULTS: Urinary alpha(2)-AP-resistant plasmin activity corrected for creatinine concentration (units/g x creatinine) was significantly higher in patients with APSGN (2.99 +/- 0.63) than in patients with IgAN (1.02 +/- 0.20, P < 0.01), SI (0.79 +/- 0.17, P < 0.01), or in healthy control subjects (0.73 +/- 0.18, P < 0.01). This tendency was confirmed by casein gel zymography. However urinary PA activity assessed by plasminogen-casein gel zymography did not differ between groups. NAPlr was detected in the urine of APSGN patients. CONCLUSIONS: We found elevated urinary plasmin activity resistant to alpha(2)-AP, which may be due to urinary excretion of NAPlr in patients with APSGN. This result supports the pathogenic role of the NAPlr-plasmin complex in the development of APSGN. Furthermore, alpha(2)-AP-resistant urinary plasmin activity may be useful as a diagnostic marker for APSGN.

Coagulation and inflammation in overt diabetic nephropathy: association with hyperhomocysteinemia.

BACKGROUND: Diabetic nephropathy, especially when advanced, is associated with high prevalence of atherosclerotic cardiovascular disease in which inflammation and coagulation may play pathogenic roles. We investigated the relationships between diabetic nephropathy and coagulation, fibrinolysis, or inflammation in patients with Type 2 diabetes. METHODS: We evaluated markers of inflammation and coagulation in 105 Type 2 diabetic patients with various grades of nephropathy and 49 healthy control subjects, in association with plasma total homocysteine (tHcy) measurements. RESULTS: Plasma tHcy concentrations were significantly higher in diabetic patients than in controls (8.96 +/- 3.04 vs. 6.92 +/- 1.36 micromol/l, P < 0.0001). Plasma concentrations of interleukin (IL)-6 were significantly higher in diabetic patients than in control subjects (P < 0.0001). In diabetic patients, plasma tHcy correlated positively with urinary albumin, fibrinogen, IL-6 and plasmin-alpha2-antiplasmin complex (PAP), while plasma tHcy correlated negatively with creatinine clearance (Ccr) and protein C activity. After adjustment for Ccr, IL-6 and protein C activity were significantly associated with plasma tHcy. Plasma tHcy concentrations were significantly higher in patients with overt albuminuria than in those with normoalbuminuria or microalbuminuria, as were plasma concentrations of fibrinogen, prothrombin F1+2, and interleukin-6. CONCLUSIONS: Diabetic nephropathy is associated with elevated markers for both coagulation and inflammation. High plasma homocysteine may be a link between diabetic nephropathy and both chronic inflammation and hypercoagulability, increasing cardiovascular risk.

Elevated levels of urine angiostatin and plasminogen/plasmin in cancer patients.

Previously, a specific angiogenesis inhibitor, angiostatin, discovered in urine and serum of tumor-bearing mice, was reported to potently block tumor growth and metastasis in animal models. Detection of angiostatin and its precursor proteins in urine from cancer patients has not been reported. Now, we report the development of an antibody-based analysis system that allows us to detect angiostatin and plasminogen/plasmin (Pgn/plasmin) in the urine of cancer patients. The detection system is a combination of a novel lysine-ELISA assay and Western immunoblot analysis using a specific antibody to human angiostatin and Pgn/plasmin. High levels of Pgn/plasmin were detected in the urine from various cancer patients, whereas healthy individuals showed relatively low levels of urine Pgn/plasmin. Of interest, angiostatin is detectable in urine samples of patients with various cancers, including acute lymphoblastic leukemia, suggesting that angiogenesis may play an important role in the development and progression of leukemia. Our data for the first time show that angiostatin and Pgn/plasmin are present at relatively high levels in the urine of human cancer patients. Detection of urine angiostatin in cancer patients helps us not only to understand the role of this angiogenesis inhibitor in cancer development and progression but also allows us to develop tools of cancer diagnosis and prognosis. Thus angiostatin has both therapeutic and diagnostic implications in cancer disease.