Claus Bistrup

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.

Improved renal function after early conversion from a calcineurin inhibitor to everolimus: a randomized trial in kidney transplantation.

In an open‐label, multicenter trial, de novo kidney transplant recipients at low to medium immunological risk were randomized at week 7 posttransplant to remain on CsA (n = 100, controls) or convert to everolimus (n = 102), both with enteric‐coated mycophenolate sodium and corticosteroids. The primary endpoint, change in measured GFR (mGFR) from week 7 to month 12, was significantly greater with everolimus than controls: 4.9 (11.8) mL/min versus 0.0 (12.9) mL/min (p = 0.012; analysis of covariance [ANCOVA]). Per protocol analysis demonstrated a more marked difference: an increase of 8.7 (11.2) mL/min with everolimus versus a decrease of 0.4 (12.0) mL/min in controls (p < 0.001; ANCOVA). There were no differences in graft or patient survival. The 12‐month incidence of biopsy‐proven acute rejection (BPAR) was 27.5% (n = 28) with everolimus and 11.0% (n = 11) in controls (p = 0.004). All but two episodes of BPAR in each group were mild. Adverse events occurred in 95.1% of everolimus patients and 90.0% controls (p = 0.19), with serious adverse events in 53.9% and 38.0%, respectively (p = 0.025). Discontinuation because of adverse events was more frequent with everolimus (25.5%) than controls (3.0%; p = 0.030). In conclusion, conversion from CsA to everolimus at week 7 after kidney transplantation was associated with a greater improvement in mGFR at month 12 versus CNI‐treated controls but discontinuations and BPAR were more frequent.

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 &ggr;-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 &mgr;mol/L; P<0.001), &agr;2-antiplasmin (1 &mgr;mol/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.

Mechanisms of renal NaCl retention in proteinuric disease

In diseases with proteinuria, for example nephrotic syndrome and pre‐eclampsia, there often are suppression of plasma renin–angiotensin–aldosterone system components, expansion of extracellular volume and avid renal sodium retention. Mechanisms of sodium retention in proteinuria are reviewed. In animal models of nephrotic syndrome, the amiloride‐sensitive epithelial sodium channel ENaC is activated while more proximal renal Na+ transporters are down‐regulated. With suppressed plasma aldosterone concentration and little change in ENaC abundance in nephrotic syndrome, the alternative modality of proteolytic activation of ENaC has been explored. Proteolysis leads to putative release of an inhibitory peptide from the extracellular domain of the γ ENaC subunit. This leads to full activation of the channel. Plasminogen has been demonstrated in urine from patients with nephrotic syndrome and pre‐eclampsia. Urine plasminogen correlates with urine albumin and is activated to plasmin within the urinary space by urokinase‐type plasminogen activator. This agrees with aberrant filtration across an injured glomerular barrier independent of the primary disease. Pure plasmin and urine samples containing plasmin activate inward current in single murine collecting duct cells. In this study, it is shown that human lymphocytes may be used to uncover the effect of urine plasmin on amiloride‐ and aprotinin‐sensitive inward currents. Data from hypertensive rat models show that protease inhibitors may attenuate blood pressure. Aberrant filtration of plasminogen and conversion within the urinary space to plasmin may activate γ ENaC proteolytically and contribute to inappropriate NaCl retention and oedema in acute proteinuric conditions and to hypertension in diseases with chronic microalbuminuria/proteinuria.

Renal function three years after early conversion from a calcineurin inhibitor to everolimus: results from a randomized trial in kidney transplantation

In a 36‐month, open‐label, multicenter trial, 202 kidney transplant recipients were randomized at week 7 post‐transplant to convert to everolimus or remain on cyclosporine: 182 were analyzed to month 36 (92 everolimus, 90 controls). Mean (SD) change in measured GFR (mGFR) from randomization to month 36 was 1.3 (14.0) ml/min with everolimus versus −1.7 (15.4) ml/min in controls (P = 0.210). In patients who remained on treatment, mean mGFR improved from randomization to month 36 by 7.9 (11.5) ml/min with everolimus (n = 37) but decreased by 1.4 (14.7) ml/min in controls (n = 62) (P = 0.001). During months 12–36, death‐censored graft survival was 100%, patient survival was 98.9% and 96.7% in the everolimus and control groups, respectively, and 13.0% and 11.1% of everolimus and control patients, respectively, experienced mild biopsy‐proven acute rejection (BPAR). Protocol biopsies in a limited number of on‐treatment patients showed similar interstitial fibrosis progression. Donor‐specific antibodies were present at month 36 in 6.3% (2/32) and 18.0% (9/50) of on‐treatment everolimus and control patients with available data (P = 0.281). During months 12‐36, adverse events were comparable, but discontinuation was more frequent with everolimus (33.7% vs. 10.0%). Conversion from cyclosporine to everolimus at 7 weeks post‐transplant was associated with a significant benefit in renal function at 3 years when everolimus was continued.

Plasma neutrophil gelatinase associated lipocalin (NGAL) is associated with kidney function in uraemic patients before and after kidney transplantation

BackgroundNeutrophil gelatinase associated lipocalin (NGAL) is a biomarker of kidney injury. We examined plasma levels of NGAL in a cohort of 57 kidney allograft recipients (Tx group, 39 ± 13 years), a uraemic group of 40 patients remaining on the waiting list (47 ± 11 years) and a control group of 14 healthy subjects matched for age, sex and body mass index (BMI). The kidney graft recipients were studied at baseline before transplantation and 3 and 12 months after transplantation and the uraemic group at baseline and after 12 months.MethodsNGAL was measured using a validated in-house Time-Resolved Immuno-flourometric assay (TRIFMA). Repeated measurements differed by < 10% and mean values were used for statistical analyses. Spearman rank order correlation analysis and the Kruskal-Wallis non-parametric test were used to evaluate the association of NGAL concentrations with clinical parameters.ResultsPlasma NGAL levels before transplantation in the Tx and uraemic groups were significantly higher than in the healthy controls (1,251 μg/L, 1,478 μg/L vs. 163 μg/L, p < 0.0001). In the Tx group NGAL concentrations were associated with serum creatinine (R = 0.51, p < 0.0001), duration of end-stage renal failure (R = 0.41, p = 0.002) and leukocyte count (R = 0.29, p < 0.026). At 3 and 12 months plasma NGAL concentrations declined to 223 μg/L and 243 μg/L, respectively and were associated with homocysteine (R = 0.39, p = 0.0051 and R = 0.47, p = 0.0007).ConclusionsPlasma NGAL is a novel marker of kidney function, which correlates to duration of end-stage renal failure (ESRD) and serum creatinine in uraemic patients awaiting kidney transplantation. Plasma NGAL is associated with homocysteine in transplanted patients. The prognostic value of these findings requires further studies.

Physiological regulation of epithelial sodium channel by proteolysis

Purpose of reviewActivation of epithelial sodium channel (ENaC) by proteolysis appears to be relevant for day-to-day physiological regulation of channel activity in kidney and other epithelial tissues. Pathophysiogical, proteolytic activation of ENaC in kidney has been demonstrated in proteinuric disease. Recent findingsA variation in sodium and potassium intake or plasma aldosterone changes the number of cleaved &agr; and &ggr;-ENaC subunits and is associated with changes in ENaC currents. The protease furin mediates intracellular cleavage, whereas the channel-activating protease prostasin (CAP-1), which is glycophosphatidylinositol-anchored to the apical cell surface, mediates important extracellular cleavage. Soluble protease activity is very low in urine under physiological conditions but rises in proteinuria. In nephrotic syndrome, the dominant soluble protease activity is plasmin, which is formed from filtered plasminogen via urokinase-type plasminogen activator. Plasmin activates ENaC directly at high concentrations and through prostasin at lower concentrations. SummaryThe discovery of serine protease-mediated activation of renal ENaC in physiological and pathophysiological conditions opens the way for new understanding of the pathogenesis of proteinuric sodium retention, which may involve plasmin and present several potential new drug targets.

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 ± 1/77 ± 0.7 mmHg; HbA1c 7.35%; and eGFR 81.0 ml/min per 1.73 m2 (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.

Aberrant glomerular filtration of urokinase-type plasminogen activator in nephrotic syndrome leads to amiloride-sensitive plasminogen activation in urine

In nephrotic syndrome, aberrant glomerular filtration of plasminogen and conversion to active plasmin in preurine are thought to activate proteolytically epithelial sodium channel (ENaC) and contribute to sodium retention and edema. The ENaC blocker amiloride is an off-target inhibitor of urokinase-type plasminogen activator (uPA) in vitro. It was hypothesized that uPA is abnormally filtered to preurine and is inhibited in urine by amiloride in nephrotic syndrome. This was tested by determination of Na(+) balance, uPA protein and activity, and amiloride concentration in urine from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Urine samples from 6 adult and 18 pediatric patients with nephrotic syndrome were analyzed for uPA activity and protein. PAN treatment induced significant proteinuria in rats which coincided with increased urine uPA protein and activity, increased urine protease activity, and total plasminogen/plasmin concentration and Na(+) retention. Amiloride (2 mg·kg(-1)·24 h(-1)) concentration in urine was in the range 10-20 μmol/l and reduced significantly urine uPA activity, plasminogen activation, protease activity, and sodium retention in PAN rats, while proteinuria was not altered. In paired urine samples, uPA protein was significantly elevated in urine from children with active nephrotic syndrome compared with remission phase. In six adult nephrotic patients, urine uPA protein and activity correlated positively with 24 h urine protein excretion. In conclusion, nephrotic syndrome is associated with aberrant filtration of uPA across the injured glomerular barrier. Amiloride inhibits urine uPA activity which attenuates plasminogen activation and urine protease activity in vivo. Urine uPA is a relevant target for amiloride in vivo.

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 ± 30%). 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 ± 1 and 16 ± 1 &mgr;mol/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.