Urinary Thrombin as a Marker of Glomerular Inflammation Associated with Renal Injury in Type 2 Diabetes.

Glomerular inflammation is a putative aggravation factor for type 2 diabetic nephropathy and urinary thrombin is a novel marker of glomerular inflammation. To clarify the relationship between glomerular inflammation and progression of the nephropathy, we measured urinary thrombin in 118 patients with type 2 diabetic nephropathy at different stages. To investigate the implications of urinary thrombin in the nephropathy, we compared urinary thrombin with expression of tissue factor, the trigger of blood coagulation activation, in glomeruli and with markers of renal injury (estimated glomerular filtration rate (eGFR) and proteinuria). Urinary thrombin was found in 4.9% (3/61), 0.0% (0/12), 29.6% (8/27) and 50.0% (9/18) of patient groups at stages 1, 2, 3 and 4, respectively. Thus, urinary thrombin was negligible in the patients at early stages (stages 1 and 2), but was present predominantly in the patients at advanced stages (stages 3 and 4). Tissue factor was expressed in accumulated macrophages in glomeruli, which indicates that thrombin may be generated in inflamed glomeruli presumably via inflammation-induced activation of the exudated coagulation factors into glomerular tissues and then be excreted in urine. Urinary thrombin was significantly associated with both decreased eGFR and increased proteinuria in type 2 diabetic nephropathy. Therefore, increased urinary thrombin in patients with advanced stages of type 2 diabetic nephropathy suggests that glomerular inflammation may injure the tissues, thereby impairing renal function. Monitoring an effect of anti-diabetic treatments on glomerular inflammation in the patients with type 2 diabetic nephropathy may be a possible application of urinary thrombin.

Competition-derived FRET-switching cationic conjugated polymer-Ir(III) complex probe for thrombin detection.

A novel, label-free and convenient strategy for thrombin assay has been developed based on the fluorescence resonance energy transfer (FRET) from a cationic conjugated polymer (CCP) to Ir(III) complex. The energy donor (CCP) and acceptor (Ir(III) complex) were taken into close proximity through π-π stacking interaction and electrostatic interaction, leading to the occurrence of FRET. However, the introduction of the thrombin aptamer upset the status and blocked the FRET process, but afterwards the reappearance of FRET phenomenon was confirmed by the special binding interaction between aptamer and thrombin, thus achieving the quantitative detection of thrombin. This assay could detect thrombin as low concentration as about 0.05pM and provided a highly specific selectivity among other nonspecific proteins. Moreover, the strategy may allow our platform to provide similar sensitivity toward different targets in an aptamer-structure-independent manner. Furthermore, the assay can be used to detect thrombin in diluted real urine or serum samples with a satisfactory recovery, implying its great potential for rapid detection of thrombin in the clinic.

Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent.

Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome.

Cleaved Form of Osteopontin in Urine as a Clinical Marker of Lupus Nephritis.

We assessed the utility of two forms of osteopontin (OPN), OPN full and its cleaved form (OPN N-half), in plasma and urine as markers of disease activity in lupus nephritis (LN). Samples were collected from patients with systemic lupus erythematosus (SLE) (LN: N = 29, non-LN: N = 27), IgA nephropathy (IgAN) (N = 14), minimal change nephrotic syndrome (MCNS) (N = 5), diabetic nephropathy (DN) (N = 14) and healthy volunteers (HC) (N = 17). While there was no significant difference in urine OPN full concentration between groups, urine OPN N-half concentration was significantly higher in patients with LN than HC (p < 0.05). Moreover, urine OPN N-half was higher in LN patients with overt proteinuria (urine protein/creatinine ratio: P/C > 0.5) than LN patients with minimal proteinuria (P/C < 0.5, p < 0.0001), and also higher than in DN patients with overt proteinuria (P/C > 0.5, p < 0.01). Urine thrombin activity correlated with urine OPN N-half concentration (p < 0.0001), but not with urine OPN full concentration. These results suggest that urine OPN N-half concentration reflects renal inflammation. Thus, urine OPN N-half may be a novel disease activity marker for LN.

Nanostructured bioluminescent sensor for rapidly detecting thrombin.

Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470 nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10 min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8 nM to 8 µM in both buffer and human urine spiked samples. The limit of detection is as low as 80 pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine.

Urinary thrombin: a novel marker of glomerular inflammation for the diagnosis of crescentic glomerulonephritis (prospective observational study).

BACKGROUND: Crescentic glomerulonephritis (CresGN), an uncommon rapidly progressive disease, is characterized by severe glomerular inflammation with fibrin deposition. The lack of specific CresGN biomarkers delays diagnosis and threatens life. Because fibrin deposits in CresGN glomeruli indicate thrombin generation, we hypothesized that thrombin is excreted in urine and is a specific CresGN biomarker. METHODS: We measured urinary thrombin activity in 200 untreated patients (17 with CresGN, 183 with primary glomerulonephritis) and controls (8 patients with healed CresGN, 11 with nephrosclerosis, and 10 with tubulointerstitial nephritis, and 66 healthy volunteers). CresGN types included 15 pauci-immune and 2 immune complex. We assessed the diagnostic accuracy of thrombinuria in 169 patients with hematuria and proteinuria. Renal biopsy tissues were immunostained for tissue factor and fibrin. We analyzed the relationship of thrombinuria to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, glomerular fibrin deposition, antineutrophil cytoplasmic antibodies (ANCAs), and C-reactive protein (CRP). We studied changes in thrombin activities after glucocorticoid treatment in 12 patients with thrombinuria. RESULTS: The highest thrombinuria occurrence was in CresGN (70.6%), followed by membranoproliferative glomerulonephritis (41.7%), IgA nephropathy (9.2%), and acute glomerulonephritis (0%). More than 75% of patients with nonproliferative glomerulonephritis manifested no thrombinuria. No controls had thrombinuria. Thrombinuria showed high CresGN specificity (90.1%) and moderate sensitivity (70.6%) and was detected in 4 of 7 patients with ANCA-negative CresGN. In CresGN, thrombinuria was associated with fibrin deposition in glomerular extracapillary tissue, where monocytes/macrophages expressed tissue factor. Thrombinuria in CresGN was unrelated to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, and CRP. After glucocorticoid treatment, thrombinuria in patients with CresGN rapidly disappeared but proteinuria and hematuria persisted. CONCLUSIONS: Thrombinuria was specific for glomerular inflammation, was unaffected by systemic inflammation or coagulation, and demonstrated good diagnostic accuracy for CresGN including ANCA-negative cases. Thrombinuria measurement may provide risk-free diagnosis and screening for CresGN.

[Evaluation of thrombin in urine as a real-time indicator of clotting activation in glomerulonephritis].

When tissues are injured and bleeding occurs, blood clotting is immediately activated and fibrin clots are formed by thrombin. Afterwards, antithrombin III promptly inactivates thrombin, which restricts the clotting to the bleeding site. In inflamed sites, tissue factor is expressed on cells in the lesion by stimulation from cytokines, and produces thrombin. In this case, thrombin may survive longer because of inefficient inactivation by antithrombin III due to dilution and less perturbation in the interstitial fluid, and therefore, has a greater chance to activate thrombin receptors (protease-activated receptors: PARs) on the cells, which induces various cellular events including proliferation, migration, and shape change. Recent studies have suggested a pathophysiological association of the PAR pathway with crescentic glomerulonephritis. However, the role of thrombin in human diseases has not been fully studied, probably because of a lack of simple and reliable methods for measuring thrombin in clinical samples. To solve this problem, we developed an ELISA system for human alpha-thrombin and applied it to the measurement of thrombin in the urine of patients with glomerulonephritis. Thrombin in urine was detected in glomerulonephritic patients but not in healthy volunteers or disseminated intravascular coagulation patients, which suggests that thrombin in urine may reflect thrombin generation by clotting activation in the glomerular lesion.

Assessment of thrombin in the urine of glomerulonephritic patients by enzyme-linked immunosorbent assay.

BACKGROUND: Accumulating evidence suggests that blood clotting occurs in inflamed glomeruli, although its role in the pathophysiology of glomerulonephritis remains to be elucidated. To address this issue, a simple and reliable method for evaluating clotting in glomeruli is necessary. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for thrombin in urine to evaluate the degree of clotting activation in diseased glomeruli. METHODS: Monoclonal antibodies against human alpha-thrombin were raised and used for sandwich ELISA to measure thrombin. Thrombin was measured in urine samples from normal volunteers and from patients with glomerulonephritis or disseminated intravascular coagulation (DIC). RESULTS: Thrombin antigen was not detected in the urine of healthy volunteers or of patients with DIC, but was detected in the urine from two-thirds of glomerulonephritic patients. The average concentration in positive samples was 3.79 microg/L. Urinary thrombin concentrations measured by ELISA correlated well with thrombin activities measured by hydrolysis of a synthetic substrate. CONCLUSION: We suggest that thrombin antigen in urine measured by ELISA is not affected by systemic thrombin production in the vessels, and reflects blood clotting activation in glomerulonephritic lesions. A close relationship between urinary thrombin and glomerulonephritis indicates a possible involvement of clotting in disease development, and measurement of urinary thrombin may provide a real-time marker for monitoring renal diseases.